The invention relates to polymer composite compositions.
Soluble collagen isolated from tissue sources such as tendon and skin forms native fibrils at 37xc2x0 C. in physiological buffers. Although these fibrils can be extruded to form synthetic fibers of various dimensions, the tensile strength of these fibers is relatively weak due to a lack of intermolecular cross-linking between collagen polypeptides. This physical weakness limits the use of these fibers in tendon and ligament reconstruction. To strengthen these collagen fibers, cross-linkers such as glutaraldehyde and carbodiimide have been used to re-establish the intermolecular cross-link. A drawback of glutaraldehyde cross-linked materials for use in vivo, however, is that glutaraldehyde and its reaction products are toxic to cells.
The invention is based on the discovery that polymeric materials, e.g., collagen, including collagen fibers, can be strengthened by adding particular catechol-containing compounds (especially compounds having two or more catechol groups) to the polymeric material and forming a polymer of the compounds that intercalate within the polymeric material, e.g., forming a polymer composite. It is believed that the resulting polymer forms a scaffold-like structure throughout the polymeric material without the necessity of cross-linking the individual polymeric materials, e.g., collagen polypeptides. This scaffolding provides synthetic polymer fibers having a tensile strength, stiffness, and strain at failure that is comparable to or better than natural polymeric material fibers.
Accordingly, the invention features a method of treating a polymeric material, e.g., collagen, by providing a mixture comprising the polymeric material and a monomer having a first catechol group; oxidizing the mixture; and polymerizing the monomer via the first catechol group to form a polymer in which the first catechol group has been oxidized to a quinone group, and the polymer intercalates into the polymeric material. The method optionally includes removing unpolymerized monomer from the mixture after the polymerizing step. The monomer can further contain a reactive group, such as a second catechol group or an aldehyde group. Alternatively, the monomer can contain, other than the first catechol group, a reactive group and a linker of at least three carbon atoms between the first catechol group and the reactive group, where no more than one peptide bond, or alternatively no peptide bond, separates the first catechol group from the reactive group. In another example, the monomer can contain a first catechol group and a reactive group, provided that the reactive group is not a carboxyl group or a primary amine. The reactive group can participate in a covalent bond with a collagen polypeptide (e.g., when the reactive group is an aldehyde, amino, or carboxyl group) or with another monomer (e.g., when the reactive group is a catechol group). When the reactive group is a second catechol group, the monomer can form a homopolymer of the monomer.
Specific examples of monomers include 2,3-dihydroxybenzaldehyde, 3,4-dihydroxybenzaldehyde, rosemarinic acid, nordihydroguaiaretic acid, and the multi-armed structures described in the Examples.
A polymeric material is any polymer that can be synthetic, natural, or derived from natural sources, e.g., marine or terrestrial animal or plant (e.g., bovine, porcine, equine, skate, or sea cucumber). The polymeric material may be in any form including solid, liquid, or gel. Polymers include, for example, collagen, gelatin (included denatured gelatin), alginates, chitosan, silk, and cellulose.
The collagen can be of any form and from any origin. For example, the collagen can be sea cucumber dermis collagen, bovine tendon collagen, molecularly engineered collagen, or gelatin (e.g., in any suitable form including hydrogels, liquids, or foams). In addition, the collagen can be digested with a protease before the oxidizing and polymerizing steps. The collagen can be in the form of microfibrils, fibrils, natural fibers, or synthetic fibers. The polymeric material, e.g., collagen, can be at least 50% (e.g., at least 75, 90, or 95%) by weight of the mixture.
In the oxidation step, oxygen can be introduced into the mixture in the form of dissolved molecular oxygen or in the form of periodate (e.g., sodium meta-periodate). The oxidation step can be carried out more rapidly by the introduction of chemical oxidants, like periodate. Oxygen introduced merely by atmospheric exposure or in vivo are suitable methods for carrying out the oxidation step. Alternatively, in areas where exposure to air is not possible or desirable, oxygen or other oxidants can be introduced from exogenous sources via, for example, tube, feed line, or cannula (e.g., arthroscopically).
In another aspect, the invention includes a method of increasing the tensile strength or the protease resistance of a composition containing collagen by adding a monomer as described above; and treating the mixture using the methods described above.
In another aspect, the invention features a composition containing a polymeric material, e.g., collagen, and a polymer that intercalates into the polymeric material, e.g., collagen, the polymer formed of monomers, each monomer having a first quinone group, a second quinone group, and a linker of at least three carbon atoms between the first quinone group and the second quinone group, where no more than one peptide bond separates the first quinone group from the second quinone group, alternatively where at least one peptide bond separates the first quinone group from the second quinone group, or alternatively where there is no peptide bond between the first quinone group and the second quinone group. Alternatively, the monomer has a quinone group and a reactive group, provided that the reactive group is not an amino or carbonyl group participating in a peptide bond within the monomer, or alternatively wherein the reactive group is an aldehyde or a second catechol.
In another aspect, the invention features a composition containing a polymeric material, e.g., collagen, and a polymer that cross-links with the polymeric material, e.g., collagen, the polymer formed of monomers, each monomer having a first quinone group, a second quinone group, and a linker of at least three carbon atoms between the first quinone group and the second quinone group, where no more than one peptide bond separates the first quinone group from the second quinone group, alternatively where at least one peptide bond separates the first quinone group from the second quinone group, or alternatively where there is no peptide bond between the first quinone group and the second quinone group; and wherein a functional group (e.g., sulfur or nitrogen or oxygen atom), from the polymeric material, e.g., collagen, chemically reacts to form a bond (either reversible or irreversible) between the monomer and the polymeric material, e.g., collagen. Alternatively, the monomer has a quinone group and a reactive group (e.g., an aldehyde or aldehyde functional equivalent, such as imine), provided that the reactive group is not an amino or carbonyl group participating in a peptide bond within the monomer.
Although the polymers of monomers described above have a first and a second quinone group, the quinone group may be reacted with another functional group in the polymer or may be cross-linked with another group in the polymeric material (e.g., collagen) to ultimately form a quinone derivative. Such quinone derivatives are deemed to be quinone groups in the polymers of monomers of the invention. For example, if a quinone group reacts with an amino group from the polymeric material, an imine (a quinone derivative) forms. It is also possible for two quinones to react, in which case a coupled quinone results. In such instances, the resulting product is considered to have two quinone groups, however, they are separated by a substituted-ethylene group formed from two of the quinone groups of the original first and second quinone.
Another aspect of the invention involves a method of making a polymer composition comprising combining collagen and a polymer that intercalates into the collagen, the polymer comprising monomers, each monomer comprising a catechol group, a reactive group (e.g., a catechol group, a quinone, an aldehyde, or aldehyde functional equivalent), and a linker of at least three carbon atoms between the catechol group and the reactive group.
In another aspect, the invention involves a method of treating a polymeric material, the method comprising: providing a mixture comprising the polymeric material and a monomer comprising a first catechol group and a reactive group selected from the group consisting of a second catechol group and an aldehyde group; oxidizing the mixture; and polymerizing the monomer via the first catechol group and the reactive group to form a polymer in which the first catechol group has been oxidized to a quinone group, wherein the polymer intercalates into the polymeric material. The catechol groups can be, for example, nordihydroguaiaretic acid, 2-Arm, 3-Arm, 4-Arm, or 9-Arm.
Another aspect of the invention involves a composition comprising a polymeric material, e.g., collagen, and a polymer made by the polymerization of a catechol-containing monomer. In one aspect, the catechol-containing monomer is a monomer comprising a first catechol group and a reactive group selected from the group consisting of a second catechol group and an aldehyde group. The catechol-containing monomer can be nordihydroguaiaretic acid, 2-Arm, 3-Arm, 4-Arm, or 9-Arm.
Another aspect of the invention is a composition comprising: a catechol-containing monomer treated collagen fiber; and a catechol-containing monomer treated collagen foam. In such compositions, the fiber can be surrounded by the foam, and can be such that the foam comprises pores of a size to allow infiltration of cells into the foam. These compositions can further comprise cells (e.g., fibroblasts, mesenchymal stem cells, chondrocytes, or molecularly engineered cells), and the catechol-containing monomer can be nordihydroguaiaretic acid, 2-Arm, 3-Arm, 4-Arm, or 9-Arm.
In another aspect, the invention involves a method of engineering tissue (e.g., tendon) comprising use of the composition comprising: a catechol-containing monomer treated collagen fiber; and a catechol-containing monomer treated collagen foam, and the variations described above.
Another aspect of the invention is a method of making a composition of the invention, comprising combining a catechol-containing monomer treated collagen fiber; and a catechol-containing monomer treated collagen foam. The combining and treatment steps can be performed in any order, thus one aspect is the method of making the composition, wherein the the catechol-containing monomer treated collagen fiber is combined with collagen foam, and the resulting composition is treated with a catechol-containing monomer.
The polymer can be a homopolymer of the monomers, each monomer attached to at least one other monomer via a covalent bond formed between a ring carbon of a quinone group of one monomer and a ring carbon of a quinone group of another monomer. The monomer can include a 2,3-dimethylbutylene group, a 1,3,5-tricarboxylic acid group, or a 5-nitroisophthalic acid group between the first quinone group and the second quinone group, or two or more additional quinone groups. As described above in regard to the methods of the invention, the source, form, and proportion of collagen in the mixture can be varied. In addition, the composition can be in the form of a synthetic fiber having a tensile strength of at least 80 MPa.
An additional aspect of the invention features a compound having three catechol groups and a linker, where each catechol group resides at a terminal carbon of the linker. Such compounds are described in the Examples below.
xe2x80x9cMicrofibrils,xe2x80x9d xe2x80x9cfibrils,xe2x80x9d xe2x80x9cfibers,xe2x80x9d and xe2x80x9cnatural fibersxe2x80x9d are the naturally-occurring structures found in a tendon. Microfibrils are about 3.5 to 50 nm in diameter. Fibrils are about 50 nm to 50 xcexcm in diameter. Natural fibers are above 50 xcexcm in diameter. A xe2x80x9csynthetic fiberxe2x80x9d refers to any fiber-like material that has been chemically or physically created or altered from its naturally-occurring state. For example, an extruded fiber of fibrils formed from a digested tendon is a synthetic fiber, but a tendon fiber newly harvested from a mammal is a natural fiber. Of course, synthetic fibers can include non-collagenous components, such as hydroxyapatite or drugs that facilitate tissue growth. For example, the compositions can contain growth factors such as basic fibroblast growth factor, tumor growth factor beta, bone morphogenic proteins, platelet-derived growth factor, and insulin-like growth factors; chemotactic factors such fibronectin and hyaluronan; and extracellular matrix molecules such as aggrecan, biglycan, and decorin.
As used herein, a xe2x80x9cterminal carbonxe2x80x9d is a carbon atom that is attached to no more than one other carbon atom within a molecule. A xe2x80x9creactive groupxe2x80x9d is a chemical moiety that facilitates formation of a covalent bond between the reactive group and (1) a catechol or quinone group, or (2) a functional group on a collagen polypeptide.
As used herein, xe2x80x9cintercalatesxe2x80x9d or xe2x80x9cintercalationxe2x80x9d is the immersion or dispersal of one substance into at least a portion of another substance.
The methods and compositions of the invention are useful in producing less immunogenic, less inflammatory, high strength, and biocompatible compositions, e.g., collagenous compositions, such as prostheses for, e.g., repair or replacement of tendons and ligaments in a mammal (e.g., a human, dog, cat, or horse). The prosthesis need not be a fiber and can be, for example, a prosthetic disk for replacing a ruptured intervertebral disk. In addition, because polymers formed from, e.g., nordihydroguaiaretic acid (NDGA), are not susceptible to protease degradation in vivo, the advantages associated with the polymer will be maintained after implantation of the collagenous composition or a device containing it inside the body.
The new compositions are also useful in tissue engineering applications. For example, the composition can include one or more fibers (e.g., collagen, collagen treated with a catechol-containing monomer described herein such as NDGA) surrounded by additional collagen material (e.g., collagen foam) such that the composition has suitable porosity such that cell infiltration can occur. In one aspect, the invention is a composition having one or more fibers surrounded by additional collagen material (e.g., collagen foam) and cells (e.g., fibroblasts, mesenchymal stem cells, chondrocytes or molecularly engineered cells) infiltrated therein. The resulting composition can be used as a skeleton or support for colonization of cells, and thus as a mechanically competent tissue engineering composition.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although suitable methods and materials for the practice or testing of the present invention are described below, other methods and materials similar or equivalent to those described herein, which are well known in the art, can also be used. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.